The present invention relates to the discovery, identification and characterization of nucleic acid molecules that encode CaSm, a novel protein that is overexpressed in various cancer tissues. The invention encompasses CaSm nucleotides, host cell expression systems, CaSm proteins, fusion proteins, polypeptides and peptides, antibodies to the gene product, antisense CaSm nucleic acids, transgenic animals that express an CaSm transgene, or recombinant knock-out animals that do not express the CaSm, and other compounds that modulate CaSm gene expression or CaSm activity that can be used for diagnosis, disease monitoring, drug screening, and/or the treatment of cancer disorders, including but not limited to cancer.
Cancer is characterized primarily by an increase in the number of abnormal cells derived from a given normal tissue, invasion of adjacent tissues by these abnormal cells, and lymphatic or blood-borne spread of malignant cells to regional lymph nodes and to distant sites (metastasis). Pre-malignant abnormal cell growth is exemplified by hyperplasia, metaplasia, or most particularly, dysplasia (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79.) The neoplastic lesion may evolve clonally and develop an increasing capacity for growth, metastasis, and heterogeneity, especially under conditions in which the neoplastic cells escape the host""s immune surveillance (Roitt, I., Brostoff, J and Kale, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps. 17.1-17.12). Clinical data and molecular biologic studies indicate that cancer is a multi-step process that begins with minor preneoplastic changes, which may under certain conditions progress to neoplasia.
Screening is the search for disease in asymptomatic people. Once an individual has a positive screening test, or signs or symptoms have been identified, further diagnostic tests are performed to determine the best course of treatment. The benefit of early detection mainly derives from the opportunity to treat disease before it has spread, when cure or control is most achievable. The American Cancer Society recommends regular cancer-related checkups for asymptomatic and at-risk individuals which include examination for cancers of the breast, colon, skin, and prostate, etc.
As understanding of the pathophysiological role of cancer increases, the role of both tumor markers and genetic information becomes more important in the management and treatment of cancer patients. Tumor markers are substances that can be measured quantitatively by biochemical or immunochemical means in tissue or body fluids to detect a cancer, to establish the extent of tumor burden before treatment, to diagnose as aides in staging or confirmation of histopathology, to predict the outcome of drug therapy, and to monitor relapse. Measurement of tumor markers have been used on screening total populations as well as in testings of high-risk groups.
Aberrant regulation of the mechanisms that control cell growth and differentiation results in cellular transformation. Molecular analysis has demonstrated that multiple mutations in oncogenies and tumor suppressor genes are required to manifest the malignant phenotype. This multi-step process is well illustrated by colorectal cancers, which typically develop over decades, and appear to require at least seven genetic events for completion (Kinzler et al., 1996, Cell, 87:159-170). Knowledge of the genetic bases of cancer has important clinical implications, the most immediate of which is improved diagnosis through genetic testing.
For example, the recent discoveries that individuals with BRCA1 and BRCA2 mutations have a predisposition to cancer may now facilitate the detection of an early onset type disease for hereditary breast cancer (Easton et al., 1993, Cancer Surv, 18:95-1131; Miki et al., 1994, Science, 266:66-71; Tavtigian et al., 1994, Nature Gen, 12:333-337). However, the incidence of these cases is just 5-10% of all known breast cancers (Easton et al., 1993, Cancer Surv, 18:95-1131; Miki et al., 1994, Science, 266:66-71; Tavtigian et al., 1994, Nature Gen, 12:333-337). Thus, early and late stage specific tumor markers are still needed for more than 90% of sporadic forms of breast malignancies.
Colorectal and breast cancers are just examples of a handful of malignant diseases which have been studied extensively at a molecular and genetic level. But there remains a large number of cancers, which awaits molecular biological characterization. The identification of tumor markers and tumor genes associated with these cancers will greatly assist in screening and identifying individuals at risk for the malignant diseases, and aid the search for novel therapeutic modalities.
Pancreatic cancer is a disease of the industrialized world, for example, the incidence in Japan has risen from 1.8 per 100,000 in 1960 to 5.2 per 100,000 in 1985. Cigarette smoking and a high fat diet have been associated with the development of the disease. (Beazley et al., 1995, Chapter 15 in Clinical Oncology, 2nd edition, ed. by Murphy et al., American Cancer Society). Ductal adenocarcinoma of the exocrine pancreas is the most common pancreatic tumor type and is the fourth leading cause of cancer deaths in the United States (Parker et al., 1996, CA-A Cancer Journal for Clinicians, 46:5-27). Cancer of the pancreas is highly malignant. Most patients are diagnosed at an advanced stage beyond the scope of potentially curative treatment (pancreatic cancer has an extremely poor prognosis with the five year survival of less than 3%; Warshaw et al., 1992, N. Engl. J. Med., 326:455-465). Distant metastases, particularly to liver, occur early in the course of the disease. Median survival after diagnosis is 6 months. An increased incidence of pancreatic carcinoma occurs among patients with chronic pancreatitis. The clinical diagnosis of pancreatic cancer is frequently made late in the course of the disease. The initial diagnostic test of choice is computed tomography (CT) scan, followed by ultrasonography. A fine needle aspiration biopsy may be obtained by CT guidance to confirm the diagnosis. The diagnostic test may provide staging information. Generally, tumor markers have not been helpful in the diagnosis or staging of pancreatic carcinoma.
Improved survival is anticipated if pancreatic cancer can be identified and detected at an early stage. Recent surgical literature reports a higher 5-year survival (up to 20%), primarily in patients with small ( less than 2 cm) tumors (Cameron et al., 1995, Surgical Clinics of North America, 75:939-951). Staging of pancreatic cancer is based upon the degree of metastasis, and patients presenting with early-stage disease have a much better prognosis than those presenting at a late stage. The majority of survivors are those who have small lesions and negative lymph nodes (T1, N0, M0).
Surgery with adjuvant therapy (5-fluorouracil and radiation) offers the best chance of success in treatment of pancreatic cancer, but unfortunately, a majority of the patients on presentation are ineligible. Treatment of unresectable cancer with drugs has been relatively disappointing even when combinations of multiple drugs are used. See Brennan et al., in Ch 27 xe2x80x9cCancer: Principles and Practice of Oncologyxe2x80x9d, 4th Ed., ed. by DeVita et al., J. B. Lippincott Co., Philadelphia, 1993.
Successful treatment, therefore, is dependent upon very early diagnosis and, thus, it is important to find additional pancreatic cancer markers that may facilitate this early detection.
Although the molecular etiology of pancreatic cancer is not defined, several genetic alterations have been detected. For example, the most common changes yet recognized are mutations in the K-ras oncogeny (Almoguera et al., 1988, Cell, 53:549-554) and mutations or homozygous deletions in several tumor suppressor genes, including TP53 (Redston et al., 1994, Cancer Res., 54:3025-3033), p16/MTS-1 (Caldas, et al., 1994, Nature Genet., 8:27-32; Huang et al., 1996, Cancer Res., 56:1137-1141) and DPC4 (Hahn et al., 1996, Science, 271:350-353). In addition, gene amplification plays a role in some pancreatic cancers (Cheng et al., 1996, Proc. Natl. Acad. Sci., USA, 93:3636-3641). However, these multiple parameters remain poorly correlated with the molecular events associated with a multi-step progression of pancreatic malignancy. Thus, there is a great need for additional genetic markers which would facilitate a better understanding of the molecular biology of pancreatic cancer, and provide the information to develop novel screening and early diagnostic tests.
The present invention relates to the identification of novel genes whose expression pattern is unregulated in cancer tissues and cell lines, and the use of such genes and gene products as targets for diagnosis, drug screening and therapies.
In particular, the compositions of the present invention encompass nucleic acid molecules that encode the novel cancer-associated Sm-like (CaSm) protein, including recombinant DNA molecules, cloned genes or degenerate variants thereof, and naturally occurring variants which encode novel CaSm gene products. The compositions of the present invention additionally include cloning vectors, including expression vectors, containing the nucleic acid molecules of the invention, and hosts which contain such nucleic acid molecules. The compositions of the present invention also encompass the CaSm gene products, variants and fragments thereof, fusion proteins, and antibodies directed against such CaSm gene products or conserved variants or fragments thereof.
The nucleic acid sequence of the human CaSm gene (SEQ ID NO: 1) is deposited with GenBank and is given the accession number AF000177. The CaSm gene produces a transcript of approximately 1.2 kb and encodes a protein of 133 amino acids with a molecular weight of approximately 15,179 daltons. Transcripts were detected in several cancer cell lines, as well as various normal tissues, including thymus, breast, colon, kidney, pancreas and heart. The amino acid sequence of the predicted full length CaSm gene product does not contain either a recognizable signal sequence or transmembrane domain, indicating that the CaSm gene product is an intracellular protein. The amino acid sequence shares significant homology with the small nuclear ribonucleoprotein (snRNP) Sm G protein.
The present invention further relates to methods for the diagnostic evaluation and prognosis of cancer, especially pancreatic cancer. For example, nucleic acid molecules of the invention can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for detection of abnormal expression of the CaSm gene.
Antibodies to CaSm gene product of the invention can be used in a diagnostic test to detect the presence of CaSm gene product in body fluids. In specific embodiments, measurement of CaSm gene product levels can be made to detect or stage cancer, especially pancreatic cancer.
The present invention also relates to methods for the identification of subjects having a predisposition to cancer. For example, nucleic acid molecules of the invention can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for the identification of CaSm gene mutations, allelic variations and regulatory defects in the CaSm gene.
Further, methods and compositions are presented for the treatment of cancer, especially pancreatic cancer. Such methods and compositions are capable of modulating the level of CaSm gene expression and/or the level of CaSm gene product activity. Inhibition of CaSm expression by antisense RNA reduced the transformed phenotype of pancreatic cancer cell lines, and the tumorigenicity of cancer cells when injected into SCID mice.
Still further, the present invention relates to methods of use of the CaSm gene and/or CaSm gene products for the identification of compounds which modulate CaSm gene expression and/or the activity of CaSm gene products. Such compounds can be used as agents to prevent and/or treat cancer. Such compounds can also be used to palliate the symptoms of the disease, and control the metastatic potential of the cancer.